In vitro evaluation of Trichoderma Harzianum and botanicals on the radial growth of Colletotrichum dematium causing anthracnose disease of groundnut (Arachis hypogaea L.)

A study was conducted in vitro to control Colletotrichum dematium causing anthracnose disease of groundnut with Trichoderma harzianum and botanicals. Five botanicals viz., Datura leaf extract, Tulsi leaf extract, Garlic bulb extract, Neem oil and Eucalyptus oil at the rate of 5% were evaluated for their efficacy against the radial colony growth of C. dematium. The complete inhibition was obtained in Eucalyptus oil (100%) followed by T. harzianum (71.01%), datura leaf extract (64.78%), tulsi leaf extract (63.63%), neem oil (49.14%) and garlic bulb extract (43.35%). In the present study different culture media viz., malt extract agar, Czapek dox agar, corn meal agar, Martin’s rose Bengal agar and oat meal agar were used for the study of different cultural characters of Colletotrichum dematium.

Original Research Article https://doi.org/10.20546/ijcmas.2019.801.279

In vitro Evaluation of Trichoderma harzianum and Botanicals on the Radial Growth of Colletotrichum dematium Causing Anthracnose

Disease of Groundnut (Arachis hypogaea L.)

Rupesh V Patil*, Shashi Tiwari and Rohan D Lokhande

Department of Plant Pathology, Naini Agricultural Institute, Sam Higginbottom University of

Agriculture, Technology and Sciences, Allahabad 211007, U.P., India

*Corresponding author

Introduction

The peanut, groundnut pea, or groundnut

(Arachis hypogaea L.) is a native of South

America but was early carried to the old world

tropics by the Portuguese explorers

Groundnut is the one of the world’s important

oilseed crops Groundnut is called as the

‘King’ of oilseeds It is one of the most

important food and cash crops in India While

being a valuable source of all the nutrients, it

is a low-priced commodity In groundnut

several diseases like tikka, rust, peanut bud necrosis, collar rot, and anthracnose are constraints the yield and productivity Anthracnose of groundnut caused by

Colletotrichum dematium was first reported by Subrahmanyam et al., (2012) The term

‘Anthracnose’ literally means ‘like coral’ and first used by Fabre and Dunal to describe a disease of grapes in which blackening of tissues was characteristic feature black lesions, usually sunken caused by certain imperfect fungi that produce conidia in acervuli those

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 01 (2019)

Journal homepage: http://www.ijcmas.com

A study was conducted in vitro to control Colletotrichum dematium causing anthracnose disease of groundnut with Trichoderma harzianum and botanicals Five botanicals viz., Datura leaf extract, Tulsi leaf extract, Garlic bulb extract, Neem oil and Eucalyptus oil at the rate of 5% were evaluated for their efficacy against the radial colony growth of C dematium The complete inhibition was obtained in Eucalyptus oil (100%) followed by T harzianum (71.01%), datura leaf

extract (64.78%), tulsi leaf extract (63.63%), neem oil (49.14%) and garlic bulb

extract (43.35%) In the present study different culture media viz., malt extract

agar, Czapek dox agar, corn meal agar, Martin’s rose Bengal agar and oat meal

agar were used for the study of different cultural characters of Colletotrichum dematium

K e y w o r d s

Anthracnose,

Colletotrichum

dematium,

Trichoderma

harzianum and

botanicals

Accepted:

17 December 2018

Available Online:

10 January 2019

Article Info

are hyaline, one-celled, that is Colletotrichum

(Jha et al., 2012) Colletotrichum dematium

until recently was a relatively poorly known

species in urgent need of epitypification It

was originally collected from a stem of

Eryngium in France as well as solanaceous

hosts and has been more recently recorded

from numerous hosts such as a pathogen of

chilli (Than et al., 2008) It has been also

recorded as a pathogen of Polygonatum

falcatum (Tomioka et al., 2008) and an

endophyte of Pteromiscum sp (Ren et al.,

2008) Disease symptoms are reported to

range from fruit rot to shoot, leaf, and flower

blight, e.g., Sutton reported that in herb It was

represented that 216 collections from 37

countries on 118 different host genera

Colletotrichum dematium is difficult to

recognize based on morphological

characteristics, mainly because different

researchers have described conidia width

differently Colonies of putative C dematium

strains have been reported by Sutton (1992) to

be very variable with white to pale

mouse-grey or mouse-grey-vinaceous patches with abundant

setae and black, conical sclerotia Conidia are

formed in olive-grey to light

vinaceous-salmon masses, and are 18–26 × 2–3 μm,

falcate, fusiform, and gradually tapered to

each end (Sutton, 1992) Appressoria are

medium brown, clavate, ovate to irregular,

margin entire or slightly irregularly lobed

(Sutton, 1992)

Keeping in view the economic importance of

anthracnose disease, the present study has

therefore been undertaken with the objective

to isolate and identify the pathogen

Colletotrichum dematium, to observe the

effect of Trichoderma harzianum and certain

botanicals on the radial growth of

Colletotrichum dematium and to study the

cultural characters of Colletotrichum

dematium on different culture media

Materials and Methods

An experiment was conducted to evaluate

effect Trichoderma harzianum and botanicals

on the radial growth of Colletotrichum dematium causing anthracnose of groundnut in vitro The experiment was conducted in the

Department of Plant Pathology, Sam Higginbottom University of Agriculture,

Technology and Sciences, Allahabad (U.P.) Isolation and identification of pathogen

Diseased leaves (anthracnose) of groundnut collected from research field of University were isolated by using standard procedure of Aneja (2004)

The pathogen was identified based on its cultural and morphological characters Following single hyphal-tip technique, the fungus was transformed/subcultured aseptically onto the PDA slant in test tubes Through frequent sub-culturing, the fungus was purified and pure culture was maintained

on agar slants in test tubes stored in refrigerator for further studies

In vitro evaluation of biological agent

Trichoderma harzianum was evaluated in-vitro on radial growth C dematium applying

Dual culture Technique (Dennis and Webster, 1971) and using Potato Dextrose Agar (PDA)

as basal culture media

In vitro evaluation of botanicals

A total of five botanicals viz Datura leaf extract, Tulsi leaf extract, Garlic bulb extract,

Neem oil and Eucalyptus oil at 5% concentration were evaluated in vitro on radial growth of C dematium applying Poison Food

Technique (Nene and Thapliyal, 1993) and using Potato Dextrose Agar (PDA) as basal culture media

Cultural characters of Colletotrichum

dematium

Different culture media viz., Malt extract agar,

Czapek dox agar, Corn meal agar, Martin’s

rose Bengal agar and Oat meal agar were used

for assessing the cultural characters such as

colony diameter, growth rate and different

phenotypic characters such as colony shape,

colony margin, colony color and substrate

color of Colletotrichum dematium

Three replications were maintained for each

media and were incubated at room

temperature and observation recorded The

different colony characters were recorded in

each medium by visual observation after 7

days of incubation

Colony diameter of every culture was

recorded daily for 7 days Growth rate was

calculated as the 7-day average of mean daily

growth (mm per day)

Collection and analysis of data

After 7 days of incubation, radial growth

(mm) of Colletotrichum dematium in

petridishes was recorded The radial growth

(mm) of mycelium of each plate was

measured by taking average of the two

diameters taken right angles for each colony

Percentage inhibition of growth was

calculated using the following formula:

Per cent growth inhibition (I) =

C - T

× 100

C

Where,

C = Growth of test fungus (mm) in control

plate

T = Growth of test fungus (mm) in treatment

plate

Results and Discussion

Identification of Colletotrichum dematium

Colony of putative C dematium was very

variable with white to pale mouse-grey or grey-vinaceous patches with abundant setae and black, conical sclerotia The conidia are borne on conidiophores, each conidia was one celled hyaline, typically long, falcate, fusiform, and gradually tapered to each end the acervuli are main distinct features of this genus that are blackish to dark brown with pointed caps, the seta are hyaline and yellowish The morphological observations of fungus were recorded by adapting slide culture technique The fungus under study was

identified as Colletotrichum dematium and its

identification results were similar to the different fungal characters given by Sutton (1992) (Fig 1)

In vitro evaluation of bioagent and

botanicals

Different treatments tested in the present study gave appreciable inhibition in radial growth of

C Dematium as shown in the Table 1

Minimum radial growth of 0.0 mm was observed in T6 (Eucalyptus oil @ 5%) which

is statistically significant followed by T1

(Trichoderma harzianum) 17 mm, T2 (Datura leaf extract @ 5%) 20.66 mm, T3 (Tulsi leaf extract @ 5%) 21.33 mm, T5 (Neem oil @ 5%) 29.83 mm and T4 (Garlic bulb extract @ 5%) 33.13 mm as compared to control (58.66 mm).Maximum per cent growth inhibition of

Colletotrichum dematium100% was obtained

by T6 (Eucalyptus oil @ 5%) followed by T1

(Trichoderma harzianum) 71.01%, T2 (Datura leaf extract @ 5%) 64.78%, T3 (Tulsi leaf extract @ 5%) 63.63%, T5 (Neem oil @ 5%) 49.14% and T4 (Garlic bulb extract @ 5%) 43.35% as compared to control (Table 1; Fig

2 and 3)

Table.1 Effect of bioagent and botanicals on the radial growth and per cent growth inhibition of

Colletotrichum dematium

T 1 Trichoderma

harzianum

Table.2 Mean colony diameter and growth rate of Colletotrichum dematium on different culture

media

diameter (mm)

Growth rate (mm/day)

Fig.1 Effect of bioagent (Trichoderma harzianum) on radial growth of Colletotrichum dematium

Fig.2 Effect of botanicals on radial growth of Colletotrichum dematium Where, A- Control, B-

Datura leaf extract (5%), C- Tulsi leaf extract (5%), D- Garlic bulb extract (5%), E- Neem oil

(5%), F- Eucalyptus oil (5%)

Fig.3 Growth of Colletotrichum dematium on different culture media

The minimum radial growth was observed in

T6(Eucalyptus oil @ 5%) whereas the

maximum radial growth was observed in T0

(Control) The probable reason for such

findings may be that the mycelial growth of

the test pathogen (Colletotrichum dematium)

was checked due to the fungicidal properties

of essential oil used during the experiment

Similar findings have been reported by

Ramezani et al., (2002)

Cultural characters of Colletotrichum

dematium on different culture media

There was significant difference among different culture media with respect to colony diameter which ranged from 48 to 89 mm The maximum mean colony diameter as observed in Czapek dox agar (89 mm) followed by Oat meal agar (73.16 mm), Malt extract agar (68.33 mm), Corn meal agar

(63.50 mm) and Martin’s rose Bengal agar

(48 mm) Colletotrichum dematium growth

rate ranges from 6.85 to 12.71 mm/day The

fastest growth was recorded 12.71 mm/day on

Czapek dox agar followed by Oat meal agar

(10.45 mm/day), Malt extract agar (9.76

mm/day), Corn meal agar (9.07 mm/day) and

Martin’s rose Bengal agar (6.85 mm/day)

(Table 2) The cultural characters and growth

of Colletotrichum dematium varied on

different media This might be due to the

variation in the nutritional requirement of the

fungus There was a wide variation in the

colony shape, margin and colour of

Colletotrichum dematium on different culture

media Similar observations were made by

Denobys and Baudry (1995), Kuramae et al.,

(1997) and Manjunath (2009)

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How to cite this article:

Rupesh V Patil, Shashi Tiwari and Rohan D Lokhande 2019 In vitro Evaluation of Trichoderma harzianum and Botanicals on the Radial Growth of Colletotrichum dematium

Int.J.Curr.Microbiol.App.Sci 8(01): 2657-2663 doi: https://doi.org/10.20546/ijcmas.2019.801.279